Glycated proteins are formed by an amadori rearrangement following non-enzymatic covalent bond formation by an aldehyde group of an aldose such as glucose (monosaccharide having a latent aldehyde group and derivatives thereof) and an amino group of the protein. Examples of protein amino groups include α-amino groups on the amino terminal and ε-amino groups on a lysine residue side chain in a protein. Known examples of glycated proteins formed in the body include glycated hemoglobin formed by the glycation of hemoglobin in blood and glycated albumin formed by the glycation of albumin.
Among these glycated proteins formed in the body, glycated hemoglobin (HbAlc) is attracting attention in diabetes and other clinical diagnostic fields as an important blood sugar control marker for diagnosing and managing the symptoms of diabetes patients. HbAlc concentration in blood reflects an average blood sugar value over a prior fixed period of time, and measured values thereof serve as important indicators in the diagnosis and management of the symptoms of diabetes.
An enzymatic method using amadoriase has been proposed as a method for rapidly and easily measuring HbAlc levels, and more specifically, consists of decomposition of HbAlc by an enzyme such as protease followed by quantification of α-fructosyl valyl histidine (to be represented by “αFVH”) or α-fructosyl valine (to be represented by “αFV”) isolated from the β-chain amino terminal thereof (see, for example, Patent Documents 1 to 7). In actuality, methods for cleaving αFV from HbAlc are considerably affected by contaminants and the like and have the problem of being unable to obtain accurate measured values, and consequently, current methods at present consist mainly of measurement of αFVH for the purpose of obtaining more accurate measured values.
Amadoriase catalyzes a reaction that forms glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide by oxidizing iminodiacetic acid or a derivative thereof (also referred to as an “amadori compound”) in the presence of oxygen.
Although amadoriase is found in bacteria, yeasts and fungi, reported examples of amadoriases that are particularly useful in the measurement of HbAlc as a result of having enzymatic activity on αFVH and/or αFV include amadoriases derived from Coniochaeta species, Eupenicillium species, Pyrenochaeta species, Arthrinium species, Curvularia species, Neocosmospora species, Cryptococcus species, Phaeosphaeria species, Aspergillus species, Emericella species, Ulocladium species, Penicillium species, Fusarium species, Achaetomiella species, Achaetomium species, Thielavia species, Chaetomium species, Gelasinospora species, Microascus species, Leptosphaeria species, Ophiobolus species, Pleospora species, Coniochaetidium species, Pichia species, Corynebacterium species, Agrobacterium species and Arthrobacter species (see, for example, Patent Documents 1 and 6 to 15 and Non-Patent Documents 1 to 11). Furthermore, among the aforementioned reported examples, amadoriase may also be referred to using expressions such as ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase or fructosyl amine oxidase depending on the literature.
Favorable thermal stability is one example of a property of amadoriase that is desirable in terms of formulation in a kit reagent for use as an enzyme for clinical diagnosis of diabetes.
Although actual measurement conditions vary according to individual strains, disclosures relating to the thermal stability of various types of amadoriase are found in the known literature. Namely, fungal amadoriase derived from Aspergillus terreus strain GP1 demonstrates residual activity of about 40% following heat treatment at 45° C. for 10 minutes (see, for example, Non-Patent Document 4). Fungal amadoriase derived from Fusarium oxysporum strain S-1F4 demonstrates residual activity of about 10% following heat treatment at 45° C. for 5 minutes (see, for example, Non-Patent Document 12). In addition, fungal amadoriase derived from Coniochaetidium savoryi strain ATCC 36547 demonstrates residual activity of about 80% following heat treatment at 37° C. for 10 minutes (see, for example, Patent Document 9). Each of the fungal amadoriases derived from Arthrinium sp. strain T06, Pyrenochaeta sp. strain YH807, Leptosphaeria nodorum strain NBRC 7480, Pleospora herbarum strain NBRC 32012 and Ophiobolus herpotrichus strain NBRC 6158 demonstrates residual activity of 80% following heat treatment at 40° C. for 30 minutes (see, for example, Patent Document 9). Fungal amadoriase derived from Neocosmospora vasinfecta strain NBRC 7590 demonstrates residual activity of 80% following heat treatment at 45° C. for 30 minutes (see, for example, Patent Document 9). Fungal amadoriase derived from Curvularia clavata strain YH923 demonstrates residual activity of 80% following heat treatment at 50° C. for 30 minutes (see, for example, Patent Document 9). Amadoriase derived from Cryptococcus neoformans lacking 34 to 39 amino acid residues of the carboxyl terminal region demonstrates residual activity of 40% following heat treatment at 45° C. for 10 minutes (see, for example, Patent Document 12). Amadoriase derived from Eupenicillium terrenum strain ATCC 18547 or Coniochaeta sp. strain NISL 9330 demonstrates residual activity of 80% or more at 45° C. for 10 minutes (see, for example, Patent Document 8).
Heat-resistant amadoriases have also been proposed that demonstrate further improved thermal stability as a result of substituting several amino acids of the aforementioned amadoriase proteins. More specifically, reported examples thereof include amadoriase derived from mutant Coniochaeta sp. strain NISL 9330, amadoriase derived from mutant Eupenicillium terrenum strain ATCC 18547, amadoriase derived from a mutant Aspergillus nidulans strain, and amadoriase derived from a mutant Phaeosphaeria strain (see, for example, Patent Documents 16 and 17). In particular, the mutant amadoriase produced by Escherichia coli strain JM109 (pKK223-3-CFP-T9) disclosed in Patent Document 16 (to be represented by “CFP-T9”) demonstrates extremely superior thermal stability in comparison with conventional amadoriases, and has been shown to maintain residual activity of 100% even after heat treatment at 50° C. for 60 minutes. In addition, the mutant amadoriase IE353-F282Y derived from Phaeosphaeria nodorum disclosed in Patent Document 17 has been shown to maintain residual activity of 92% following heat treatment at 50° C. for 10 minutes.
However, in the case of presuming a kit incorporating an enzyme being subjected to increasingly severe temperature conditions such as during distribution at ambient temperatures or long-distance transport, or in the case of considering applications such as an enzyme sensor which are presumed to be subjected to heat treatment in a manufacturing process, there continues to be a strong demand for enzymes having even more superior heat resistance and further improved heat resistance than the amadoriases that have been proposed thus far. Such heat-resistant enzymes are expected to make a significant contribution in the fields of enzyme and enzyme kit distribution as well as the development of sensors and other applications.